Document Type

Theses, Masters

Disciplines

1.4 CHEMICAL SCIENCES, 1.6 BIOLOGICAL SCIENCES

Publication Details

A thesis submitted in fulfilment of the requirements for the award of Master of Philosophy (MPhil)

Abstract

Probiotics are live microorganisms that when administered in adequate amounts confer a health benefit on the host. Beneficial microbes are found in several sites throughout the body such as the oral cavity, skin, respiratory tract, vagina, urinary tract, breast tissue and the gut. In this research, the culture Bifidobacterium longum ITT 13, which was previously isolated from a neonatal faecal sample, was examined for its potential as a probiotic candidate. Probiotics can have numerous desirable characteristics. The pertinent ones to this research are being of human or animal origin, not prone to antibiotic resistance, demonstration of antagonistic activity towards pathogens, absence of virulence factors and have its identification confirmed based on its genetic profile to eliminate any ambiguity.

When grown as a pure culture under anaerobic conditions at 37o C, without agitation, B. longum ITT 13 had a specific growth rate of 0.422 hr-1 . The addition to the culture medium of selected prebiotics namely starch, inulin, raffinose and fructooligosaccharide (FOS) at various concentrations, significantly altered the specific growth rate, with the addition of 0.1 % (w/v) starch resulting in the highest specific growth rate at 0.762 hr-1 . In separate experiments, B. longum ITT 13 was co-cultured with selected lactic acid bacteria (LAB), including L. acidophilus, L. lactis, L. casei and S. thermophilus. Of these organisms tested, co-culturing with L. acidophilus was found to produce the highest specific growth rate of 0.948 hr-1 as compared to 0.939 hr-1 for L. lactis, 0.576 hr-1 for S. thermophilus and 0.281 hr-1 for L. casei. This information may be helpful in scaling up and maximising the industrial biomass yield of the organism.

In a further set of experiments, the antimicrobial activity from the cell-free spent medium (CFSM) of B. longum ITT 13 against the pathogens Clostridium difficile and Salmonella Typhimurium was investigated. In the absence of prebiotics, B. longum ITT 13 CFSM did not show antimicrobial activity against these organisms. However, when grown with

the addition of the prebiotics inulin, raffinose and FOS to the culture medium, the CFSM showed promising antimicrobial activity against these two organisms. The antimicrobial activity against pathogenic Streptococcus pneumonia when B. longum ITT 13 was co-cultured with Lactobacillus acidophilus yielded a zone of clearance 17.9 mm in diameter. Antimicrobial activity against S. pneumoniae was not found in this research with the addition of any of the four prebiotics or co-culture of L. lactis, L. casei or S. thermophilus with B. longum ITT 13.

Biofilm production by bacteria is considered to be a virulence factor and has implications in pathogenicity. However, biofilm production can also allow so-called ‘good’ organisms to colonise the gut. Throughout these studies examining the ability of B. longum ITT 13 to produce a biofilm, the organism consistently failed to give positive results in tests employing Congo Red agar (CRA) plates and 96-well plates using crystal violet staining, indicating its inability to produce a biofilm. Furthermore, during a time-course study over a period of six days, the ability of B. longum ITT 13 to prevent the production of biofilms by indicator organisms was examined. When B. longum ITT 13 was co-cultured with indicator pathogens Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis and Escherichia coli, it was found that all required an adjustment period after which the level of biofilm production stabilised and large fluctuations in percentage biofilm were not observed. For example, E. faecalis yielded a biofilm of 16.8 % after 1 day incubation with this increasing to 63.2 % after 2 days but thereafter stabilising around 80 % biofilm for 3 to 6 days. Further studies in which the ability of B. longum ITT 13 to disrupt pre-existing biofilms indicate a decrease in the percentage of biofilm formed after 24 hours for each indicator organism tested. This could have implications with respect to using B. longum ITT 13 in treating but not preventing infections caused by biofilm producing pathogens.

An antibiotic resistance profile was drafted using the Kirby Bauer Disk Diffusion Susceptibility Test. Of the eighteen antibiotics tested, B. longum ITT 13 was susceptible to eleven, created intermediate zones (14 mm -15 mm) against two and was resistant to five.

The minimum inhibitory concentration (MIC) test was used to ascertain if addition of B. longum ITT 13 to a mix containing ampicillin or polymyxin B, enhanced the antibiotic’s action against two selected indicator pathogens (E. coli and S. aureus). The results were encouraging in the test with ampicillin, E. coli and B. longum ITT 13, where there was an overall reduction of E. coli bioburden relative to the effect of ampicillin on its own against E. coli. However, with increasing ampicillin concentration in combination with S. aureus and B. longum ITT 13, there appeared to be an enhancement in the growth of S. aureus, indicative of ‘Eagle Effect’, where bacteria have an increased ability to grow in the presence of antibiotic concentrations deemed to be higher than bactericidal concentrations. Co-culture of E. coli with B. longum ITT 13 in the presence of polymyxin B also did not demonstrate a positive synergistic effect.

Lastly, to confirm the identity of the test isolate, B. longum ITT 13, and satisfy the speciation requirement for a culture to qualify as a probiotic, next-generation sequencing (NGS) was performed using the Illumina MiSeq FGx which confirmed B. longum subsp. infantis speciation. The culture was aligned with a National Centre for Biotechnology Information (NCBI) reference strain using the bioinformatics software, Galaxy. This software annotated twenty genes in the B. longum ITT 13 genome and several hypothetical proteins which warrant further investigation. No antimicrobial or virulence genes were identified.

The culmination of all results in this research lead to a potentially promising future for B. longum ITT 13 to be classified as a probiotic

DOI

https://doi.org/10.21427/yxaw-am75

Creative Commons License

Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

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